Sialic acids represent a number of complex higher saccharides, most of which are terminally bound to glycoproteins and glycolipids of various tissues of animals. Sialic acids which are .alpha.-linked to glycolipids and glycoproteins that occur on cell surface, have a major role in cellular recognition processes. (See R. Schauer, Adv. Carbohydr. Chem. Biochem. (1982) 40:131-134, and R. Schauer, Cell Biology Monographs, Vol. 10 (1982) Springer-Verlag, N.Y., Wien.)
In the case of infection by influenza virus, the specific interaction between the hemagglutinin (HA, one of the major glycoproteins on the surface of the virus) with sialic acid in its .alpha.-conformer is the only known phenomenon by which the attachment of the virus to the host cell occurs, prior to its entry and proliferation within the cell. (See D. C. Wiley et al., Ann. Biochem. (1987) 56:365-394, and D. C. Wiley in Virology. B. N. Fields, ed., (1985) pp. 45-68, New York.) The receptor site on HA is the extracellular globular domain. (See I. A. Wilson et al., Nature (1981) 289:366-373, and D. C. Wiley et al., Nature (1981) 289:373-378.) It is trimeric, consisting of three conserved pockets of amino acids. Therefore, in every case of infection the receptor on the virus is this globular domain whose binding to the host cell is mediated through .alpha.-sialic acid on the cell surface. The exact interaction that occurs, involving various amino acids in the pocket with the functionalities on the sialic acid has been determined by X-ray crystallographic studies as shown in FIGS. 1 and 2 and described in W. Weis et al., Nature (1982) 333:131-234.
There are thirteen antigenically distinct HAs that have been demonstrated in human, swine, avian and equine isolates (see Hinshaw et al. in Basic and Applied Research (A. S. Bear, ed.) 1982, vol. 2, pp. 1082-96, Elsovier, Amsterdam). Thus, whereas some strains are SA.alpha.(2,3)Gal specific, the other would recognize SA.alpha.(2,6)Gal on the cell surface. In addition, some HAs would recognize 4-O-acetyl-N-acetyl neuraminic acid, a sialic acid characteristic of the equine species (H. P. Schauer et al., Biochem. Soc. Symp. (1974) 131:394-400). These exhibited variations have been shown to be species specific. Influenza strains that infect human cells recognize SA.alpha.(2,6)Gal.
A major outcome of these studies is the observation that sialic acid residues are recognized only in their .alpha.-anomeric configurations. Whereas the .alpha.-anomer is more abundant in nature, chemical reactions with sialic acid has shown that in solution, during glycosidation, the .beta.-anomer forms preponderantly, indicating it to be the thermodynamically preferred structure. Synthesis of oligosaccharides containing .alpha.-sialosides as the terminal (non-reducing end) moiety have been a challenge to carbohydrate chemists. Attempts to prepare such molecules generally involved methods commonly utilized for glycosidation reactions. Synthesis of .alpha.-sialosides have been done using methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-3,5-di-deoxy-D-glycero-D-galac to-2-nonulopyranosonate (which compound is shown in Reaction Scheme 2 below as compound 1) as the donor under Knoenigs Knor conditions (see Koenig et al., Chem. Ber. (1901) 34:957) or modification thereof using silver or mercury salts (R. Schauer et al. (eds.), "Proceedings of the Japanese-German Symposium on Sialic Acid" May 18-21, 1988; T. Ogawa et al., Carbohydr. Res. (1985) 135 C5-C9; H. Ijima et al., Carbohydr. Res. (1989) 186:107-118; V. Pozsgay et al., J. Carbohydr. Chem. (1987) 6:41-55; D. J. M. van der Vleugel et al., Carbohydr. Res. (1982) 102:121-130; N. Bagett et al., Carbohydr. Res. (1982) 110:11-18). The resulting products have been found to be largely contaminated with the .beta.-linked isomer.
Thioglycosides as the glycosyl donors have been extensively used in oligosaccharide synthesis. Their increasing importance for glycosylation reaction is mainly due to a number of thiophilic activators that have been introduced for direct synthesis of .alpha.- and .beta.-glycosides from the corresponding thioglycosides. (See F. Dasgupta et al., Carbohydr. Res.. UK 4089 (1990) 202, 229-238; F. Dasqupta et al., Carbohydr. Res: (1988) 177, C13-C17; P. Fugedi et al., Carbohydr. Res. (1986) C6-C10; P. Fugedi et al., Glycoconjugate J. (1987) 4:97-108; Y. Ito et al., Tetrahedron Lett. (1988) 29:1061-1069; Y. Ito et al., Tetrahedron Lett. (1988) 29:4701-4706; V. Pozsgay et al., J. Org. Chem. (1987) 52:4635-4637; and H. Lonn, Carbohydr. Res. (1985) 139:105-113, 115-121.
For the preparation of .alpha.-sialosides, most consistent results have been obtained using methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-di-deoxy-2-methyl-2-thio-D-glycero- .alpha.-D-galacto-2-nonulopyranosonate in conjunction with dimethyl (methylthio) sulfonium trifluoromethane sulfonate (DMTST) as the thiophilic promoter (Fugedi et al. Carbohydr. Res. (1986) C6-C10). This method of coupling has been extensively used by Kiso and Hasegawa and has proved to be of value for the synthesis of .alpha.-sialoside containing oligosaccharides. (See A Hasegawa et al., J. Carbohydr. Chem. (1986) 5:11-19.)
However, synthesis of this .alpha.-thiosialoside required a number of steps starting from compound 1 (shown in FIG. 3) and also used the carcinogenic reagent, methyl iodide (A. Hasegawa et al., J. Carbohydr. Chem. (1986) 5:11-19; O. Kanie et al., J. Carbohydr. Chem. (1988) 7:501-506). In addition, the method could not be of general use, e.g., it is not possible to make by this method .alpha.-phenyl thiosialoside, an equally effective and sometimes a thioglycoside of choice during sialosidation reaction.
The most prominent sialic acid is N-acetylneuramic acid, which occurs mainly .alpha., 2.fwdarw.3 or .alpha., 2.fwdarw.6-glycosyldically linked to the preterminal sugars. Specific synthesis methods for producing N-acetylneuramic acid and the glycal derivatives is described by E. Kerchner et al., J. Carbohydr. Chem. (1988) 10(2), 453-486, which is incorporated herein by reference. This publication specifically teaches the use of potassium methoxide as a catalyst to promote the formation of the .alpha.- as opposed to the .beta.-thioglycoside.
The present invention endeavors to solve the synthesis problems of the prior art by reducing the reaction steps needed and/or improving the yield of the .alpha.-configuration obtained.